Stem cells self-renew and produce daughters that generally proliferate before differentiating into a variety of cell types. These properties allow a single stem cell to produce sufficient progeny to maintain adult tissues. However, stem cell proliferation must be strictly limited according to need and must occur only in an environment that allows stem cell progeny to develop appropriately. Hence, stem cells are normally supported only in restricted, appropriately positioned micro-environments, termed niches. Most stem cell niches are hard to access or define. The Drosophila ovary, in which germline and somatic stem cell progeny collaborate to produce eggs, provides an exception that also affords the critical attribute of extensive and rapid investigation using molecular genetics. Here, somatic follicle stem cells (FSCs) in Drosophila ovaries will be used to ascertain the mechanisms by which a niche regulates stem cell behavior. Many types of stem cell niche interactions are found in nature, so insights gained from FSCs will undoubtedly both complement and strengthen those derived from other model stem cells. Particularly interesting aspects of the FSC model are regulation by multiple signaling pathways, competition between stem cells for niche positions and extensive regulation of niche adhesion. FSC dynamics and the impact of specific mutations also provide an excellent model for the retention and immortalization of pre-cancerous mutations that arise in stem cells. Execution of the first genetic screen for stem cell function in vivo, coupled with extensive testing of the role of signaling pathways on FSCs has provided key reagents, methods and hypotheses concerning stem cell function. This proposal aims to determine how individual signaling pathways affect FSCs and how multiple pathways are integrated, particularly to regulate niche adhesion. A very successful genetic screen will be extended to include more of the Drosophila genome and key mutations identified will be used to understand the fundamental circuits affecting FSC biology. This includes testing specific hypotheses that quality control uses enhanced checkpoint responses to DNA replication stress and co-ordinated regulation of the cell cycle and niche retention. FSC mutations that induce ectopic FSC duplication or displacement of neighboring FSCs provide critical reagents and impetus for understanding how stem cells compete for limited niche positions. New competition assays, FSC mutants derived from screens and signaling pathway studies will be supplemented by a new selection scheme to isolate mutations conferring enhanced competition in order to decipher both principles and players. Collectively, these studies should provide a pioneering, open-ended approach to building a comprehensive framework for the regulatory circuits that control stem cell behavior. PUBLIC HEALTH RELEVANCE: Stem cells are key vehicles for future regenerative therapies and are critical cells for the earliest steps of development of many cancers, yet very little are known about how stem cells are regulated because they are very difficult to identify and manipulate in their normal physiological setting. We propose extensive molecular genetic analyses of Drosophila ovarian follicle stem cells, which can be studied in great detail in their normal environment. Our findings will uncover basic principles of stem cell regulation that are directly relevant and of critical significance to our understanding of how to harness human stem cells in medicine and how to combat cancer.